Retinal development, function, and disease are, to a significant degree, controlled by the pattern of genes expressed by the cells of the retina. In an effort to better understand the mechanisms regulating photoreceptor gene expression, we have been studying rod and cone opsin gene regulation as model systems. Using a variety of approaches, we, and others, have defined some of the DNA elements important for rod- and cone-specific expression, have identified and cloned some of the transcription factors that bind to these DNA elements, and have shown that mutations in some of these factors can both interfere with normal photoreceptor development in the mouse and can cause retinal degeneration in man. This application for continued funding of these studies proposes to continue and broaden this work. The proposed work includes four aims: 1) more fully characterize several already cloned factors that are candidate molecules for regulating the various cone opsins; 2) further develop and utilize in vitro retinal electroporation technology so as to assess the biological activity of rod and cone opsin regulatory elements in a more physiological milieu, and in a complementary fashion use a high-throughput PCR-based DNase I hypersensitivity assay to identify regions of regulatory interest; 3) utilize a high- throughput transient transfection-based assay together with libraries of transcription factor expression clones to identify factors that regulate the response elements identified in 2; and 4) use a combined bioinformatic and experimental approach to identify genes that are differentially expressed in the retina, and then determine how expression of these retina-specific splice isoforms varies during development, during degeneration in the rd1 mouse, and in three mouse models that express altered forms of three pre-RNA processing factors (PRPF 3, 8 and 31) that when altered in humans cause retinitis pigmentosa. Together, these studies will hopefully provide new insights into the mechanisms regulating photoreceptor gene expression, which in turn will aid in understanding how abnormalities in gene expression can cause retinal disease, and help in the development of more cell-type specific gene therapy approaches.